Hepatocytes reprogram liver macrophages involving control of TGF-β activation, influencing liver regeneration and injury

Background: Macrophages play an important role in maintaining liver homeostasis and regeneration. However, it is not clear to what extent the different macrophage populations of the liver differ in terms of their activation state and which other liver cell populations may play a role in regulating the same. Methods: Reverse transcription PCR, flow cytometry, transcriptome, proteome, secretome, single cell analysis, and immunohistochemical methods were used to study changes in gene expression as well as the activation state of macrophages in vitro and in vivo under homeostatic conditions and after partial hepatectomy. Results: We show that F4/80+/CD11bhi/CD14hi macrophages of the liver are recruited in a C-C motif chemokine receptor (CCR2)–dependent manner and exhibit an activation state that differs substantially from that of the other liver macrophage populations, which can be distinguished on the basis of CD11b and CD14 expressions. Thereby, primary hepatocytes are capable of creating an environment in vitro that elicits the same specific activation state in bone marrow–derived macrophages as observed in F4/80+/CD11bhi/CD14hi liver macrophages in vivo. Subsequent analyses, including studies in mice with a myeloid cell–specific deletion of the TGF-β type II receptor, suggest that the availability of activated TGF-β and its downregulation by a hepatocyte-conditioned milieu are critical. Reduction of TGF-βRII-mediated signal transduction in myeloid cells leads to upregulation of IL-6, IL-10, and SIGLEC1 expression, a hallmark of the activation state of F4/80+/CD11bhi/CD14hi macrophages, and enhances liver regeneration. Conclusions: The availability of activated TGF-β determines the activation state of specific macrophage populations in the liver, and the observed rapid transient activation of TGF-β may represent an important regulatory mechanism in the early phase of liver regeneration in this context.


Supplemental Materials & Methods
Animals -C57BL/6J wt mice from Janvier, CCR2 -/and TGF-RII-loxP mice from Jackson, breed with LysM-cre mice on a C57BL/6 background, were used for the experiments. The TGF-RII-loxP mice were generated and described by Levéen et al. 2002 [1], the CCR -/mice by Boring et al. 1997 [2]. Animal housing and all the experimental procedures were reviewed and approved by the North Rhine-Westphalian State Agency for Nature, Environment, and The liver was removed from the animal and transferred into a tube with William´s medium E (Biochrom) supplemented with 10 % fetal calf serum (FCS, Biochrom), 2 mM glutamine and 1 % penicillin/streptomycin (Gibco). In a sterile hood the capsule of the liver was carefully opened by a tweezers and the liver cells were flushing out by gentle shaking. The cell suspension was placed on a 70 µm cell strainer to remove tissue residues. The cells were washed once with William´s medium, then twice with autoMACS rinsing solution supplemented with 0.5 % [w/v] BSA for separation by centrifugation (50 x g, 3 minutes, 20°C). After removing the last supernatant, antibodies against CD11b coupled to magnetic beats (Miltenyi Biotec, #130-049-601) were added to remove CD11b positive cells and incubated for 15 min at 4°C. The cell suspension was filled up to 10 ml by autoMACS rinsing solution and put on large cell separation columns (Miltenyi Biotec, #130-042-202) in a magnetic field (Fig. S1B,C). The cell number of vital hepatocytes in the flow through was determined by staining an aliquot with trypan blue.
The hepatocytes were cultivated in companion 6-well plates (BD Biosciences) with 0.8 Mio cells per well. To cultivate hepatocytes over several days under steroid-free conditions without the fibroblastoid dedifferentiation typical for the epithelial-mesenchymal transition of these cells, hepatocytes were embedded in a collagen matrix (Fig. S1F) [3]. Briefly, the plates were pre-coated with 350 µl collagen from rat tail (1 mg/ml collagen, Roche, #11179179001), In comparison to the sandwich, culture (Fig. S1F), the cells were also cultivated as monolayers. Therefore, cells were plated on rat tail collagen I-coated tissue culture dishes with William's medium E supplemented with 10 % [v/v] FCS, 100 nM dexamethasone, 2 mM L-glutamine and 1 % [v/v] penicillin/streptomycin. Cells were kept in a humidified atmosphere at 37 and 5 % CO 2 for 3 h to allow hepatocytes to attach. The cells were washed twice and cultured in medium without FCS (see above). Following incubation with this medium overnight, the experiments were started in starvation medium (William´s medium E supplemented with 2 mM L-glutamine and 1 % [v/v] penicillin/streptomycin). FcX Plus (anti-mouse CD16/32, BioLegend) and incubated for 10 minutes in the dark on ice before the corresponding antibodies in sevenfold panels (antibodies are listed in Table S3A) were added and stained overnight at 4°C. After washing twice, F4/80 positive liver cells were identified by subtracting the corresponding fluorescence minus one (FMO) control by means of flow cytometry (FACS Canto II, BD Biosciences). Further different subpopulations were defined at the expression level of CD11b and CD14 and their respective fluorescence intensity of different markers was analyzed using the gating strategy delineated in Figure S2.

Isolation of non-parenchymal liver cells and analysis by flow cytometry -
Unstained, single color and FMO control tubes were used to generate compensation matrices that correct for spectral overlap. All data were analyzed by Flow Jo 10.6.7. Co-culture -One day after preparation of primary hepatocytes and splitting BMDM the supernatant of hepatocytes and from control wells (no hepatocytes, only collagen) was removed. Per well 2 ml starvation medium was added. The supernatant of the transwells was also removed and the transwells were transferred to the 6-well plates containing hepatocytes or to control wells. Per transwell 2 ml starvation medium containing 10 ng/ml mM-CSF was added.

Isolation and differentiation of bone-marrow cells
For FACS-analysis BMDM were mono-or co-cultivated for up to four days. For stimulation with TGF-1 (5 ng/ml) it was given once, and cells were cultured for three days. Anti-TGF-1, 2, 3 (0.25 µg/ml) was given daily for three days or cells were stimulated with ECM1 For basal mRNA expression analysis BMDM were mono-or co-cultivated for one to three days. For LPS-stimulation, the cells were cultivated for two days and then stimulated with 10 ng/ml LPS for indicated time points. For RNA isolation, the transwells were put into a new 6-well plate, washed twice with PBS and lysed by RLT-buffer. prepared for mass spectrometric analysis essentially as described earlier [6]. Briefly, 5 µg of proteins per sample were shortly stacked in a polyacrylamide gel (about 5 mm running distance), subjected to silver-staining, reduced with dithiothreitol, alkylated with iodoacetamide and finally digested with trypsin.

RNA isolation, cDNA synthesis and real-time PCR
Resulting peptides were extracted from the gel, vacuum-dried and 500 ng per sample reconstituted in 0.1 % trifluoroacetic acid for subsequent liquid chromatography coupled mass spectrometric analysis.
First, an Ultimate 3000 Rapid Separation liquid chromatography system (Thermo Scientific, Dreieich, Germany) was used for peptide separation. After pre-concentration of peptides on a trap column (Acclaim PepMap100, 3 µm C18 particle size, pore size 100 Å, inner diameter 75 µm, 2 cm length, Thermo Scientific, Dreieich, Germany) for ten minutes at a flow rate of 6 ml/min using 0.1 % [v/v] TFA as mobile phase, peptides were separated on an analytical column (Acclaim PepMapRSLC, 2 µm C18 particle size, 100 Å pore size, 75 µm inner diameter, 25 cm length, Thermo Scientific, Dreieich, Germany) at 60°C using a 2 h gradient Three different sets of searches were carried out: First hepatocytes (mono and co-culture), second macrophages (mono and co-culture) and third hepatocytes and macrophages (each mono and co-culture).
For the volcano plots quantitative data was further analyzed using MSPypeline [7] and pvalues, from a pairwise comparison of two groups, were determined using the R limma package. Calculations were corrected for the intensity-variance relationship.
The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE [8] partner repository with the dataset identifier PXD012136.
Microarray Assays -The totalRNA samples used for transcriptome analyses were quantified and quality measured by capillary electrophoresis Bioanalyzer assay (Eukaryote Total RNA Pico, Agilent). All samples in this study showed high quality RNA Integrity Numbers (RIN; mean = 9.7). Synthesis of cDNA and subsequent biotin labeling of cRNA was performed according to the manufacturer's protocol (GeneChip® Pico Reagent Kit 703308 Rev. 4; ThermoFisher scientific). Briefly, 1 ng of total RNA were converted to cDNA, amplified to complementary RNA (cRNA) followed by in vitro transcription and biotin labeling of cDNA.
After fragmentation labeled cDNA was hybridized to Applied Biosystems™ Clariom™ S Mouse Gene Expression Microarrays for 16 hr at 45°C, stained by streptavidin/phycoerythrin conjugate, and scanned as described in the manufacturer's protocol.
Data analyses on Affymetrix CEL files were conducted with GeneSpring GX software (Vers.

12.5; Agilent Technologies). Probes within each probe set were summarized by GeneSprings'
ExonRMA16 algorithm after quantile normalization of probe-level signal intensities across all samples to reduce interarray variability [9]. Input data pre-processing was concluded by baseline transformation to the median of all samples. After grouping of samples (five biological replicates each) according to their respective experimental condition, a given probe set had to be expressed above background (i.e., fluorescence signal of that probe set was

Processing of 10X Genomics single cell data -
Raw sequencing data was processed using the 10X Genomics Cell Ranger software (v6.0.0). Raw BCL-files were demultipexed and processed to Fastq-files using the CellRanger mkfastq pipeline. Alignment of reads to the mm10 genome and UMI counting was performed via the CellRanger count pipieline to generate a gene-barcode matrix. All samples were aggregated and normalized for sequencing depth using the cellranger aggr pipeline. Further analyses were carried out with the Seurat v4.0.5 R package [10][11][12]. Initial quality control was performed for each sample and consisted of removal of cells with less than 200 detected genes and less than 500 unique molecular identifies. Additionally, cells belonging to the top 1 % based on the number of genes were discarded and genes expressed in less than 3 cells were removed. Furthermore, cells with a mapping rate of > 10 % to the mitochondrial genome were removed, as they represent dead or damaged cells. Cell doublets were removed from the dataset using DoubletFinder v2.0 with default parameters [13]. Normalization has been carried out using the method LogNormalize in Seurat. Samples were then integrated by selecting the top 1,500 most recurrent variable genes across all samples. PCA correction was performed with harmony [14] using sample and mouse type labels as covariates. Dimensional reduction of the data set was achieved by Principal Component analysis (PCA) based on the 3,000 most variable features and subsequent uniform manifold approximation and projection (UMAP) embedding using the first 30 principal components (PCs). With the same PCs a shared nearest neighbour graph was build using FindNeighbors and cells were clustered using the Louvain algorithm implemented in Seurat. Markers defining each cluster as well as differential gene expression between different clusters were calculated using a Wilcoxon Rank Sum test implemented in Seurat.
Finally, cells were assigned to major cell lineages by manually inspecting cluster marker genes and confirmed by performing an enrichment analysis using hypergeometric tests with cell type markers from Aizarini et al. [15]. RNA-seq data have been deposited in the ArrayExpress database at EMBL-EBI (www.ebi.ac.uk/arrayexpress) under accession number E-MTAB-11727.
Partial hepatectomy -2 / 3 PHx was performed as described previously [5]. The statistical analysis of the mass spectrometric analysis, the microarray assay and the single cell analysis is described in detail in the section of each method.    mRNA expression levels between mono-(grey) and with hepatocytes co-cultivated (black) BMDM after one to three days of cultivation. The data are presented as means ± SEM (number of replications are shown in the picture). Significant differences between mono-and co-culture are indicated by * for p ≤ 0.05, ** for p ≤ 0.01 and *** for p ≤ 0.001 (Mann-Whitney U test). The data are presented as means ± SEM (n=5-7). Significant differences between mono-and co-culture are indicated by * for p ≤ 0.05, ** for p ≤ 0.01 and *** for p ≤ 0.001 (Mann-Whitney U test).  presented as means ± SEM. Significant differences between mono-and co-culture are indicated by * for p ≤ 0.05, ** for p ≤ 0.01 and *** for p ≤ 0.001 (Mann-Whitney U test).